I. Field Of The Invention
This invention relates to an improved method for the microbial degradation of trichloroethylene by Pseudomonas mendocina KR-1 (PmKRl) or Pseudomonas putida KT2440 containing the pAUTl plasmid from PmKRl (Pp Y2101) or by genetically-engineered microorganisms containing the PmKRl toluene monooxygenase genes. It has now been unexpectedly found that the PmKRl toluene monooxygenase enzyme system is useful in the degradation of trichloroethylene. The isolation and cloning of gene segments encoding the toluene monooxygenase enzyme system of PmKRl is described in U.S. patent application Ser. No. 177,631, filed Apr. 5, 1988, which is hereby incorporated by reference.
In one aspect, the present invention relates to a method for the microbial degradation of trichloroethylene by treating trichloroethylene with a microorganism host cell containing a recombinant plasmid, the recombinant plasmid containing the PmKRl toluene monooxygenase genes. The microorganism host cell containing the recombinant plasmid must be treated with an inducer of the toluene monoxygenase genes in order to degrade trichloroethylene. The present invention provides a novel method of degrading trichloroethylene by providing genetically engineered microorganisms that exhibit levels of toluene monooxygenase enzyme activity under certain cell culture and assay conditions that exceed levels expressed in wildtype PmKRl cells. The present invention therefore provides a more efficient means of conducting certain biodegradations dependent on this enzyme system, particularly the degradation of trichloroethylene. The invention is applicable for degrading trichloroethylene as it may be present as a pollutant or contaminant in open or closed environmental systems.
Trichloroethylene (TCE) is a widely used industrial solvent that is found frequently as a contaminant in groundwater, drinking water and waste water. Groundwater is the major reservoir of improperly treated hazardous wastes. More than 200 organic and inorganic chemicals have been identified in various groundwater supplies, however, of all such identified contaminating chemicals, the EPA has identified TCE as the most frequently observed chemical contaminant at National Priority List (NPL) sites in the United States.
The magnitude of the problem of groundwater contamination is further exemplified by the fact that ground water supplies 25% of all water used in the United States. Calculations by The Conservation Foundation ("Groundwater - Saving the Unseen Resource," November, 1985 at p. 5) show that in the United States, groundwater is the source of: (1) 35% of all municipal water, (2) 50% of all drinking water (97% in rural areas), (3) 40% of all water used in agricultural irrigation, and (4) 26% of all water used by industry (excluding electric power plants). Thus, the importance of developing environmentally effective techniques for the degradation of TCE into innocuous materials cannot be overemphasized.
Development of genetically engineered microorganisms which have superior abilities to degrade specific chemical contaminants, such as TCE, to innocuous materials is one important strategy in the development of cost effective and environmentally sound methods for hazardous chemical work cleanup. The development and use of microorganism host cells with recombinant plasmids containing PmKRl toluene monooxygenase genes in the present invention is the first method employing such genetically engineered microorganisms useful for TCE chemical waste cleanup. The development and use of microorganism host cells containing the recombinant plasmids described herein are particularly advantageous because specific and well-characterized gene segments encoding PmKRl toluene monooxygenase genes been cloned and used to construct the recombinant plasmids, as described in the above referenced U.S. patent application Ser. No 177,631. It is these gene segments placed under the regulation of certain promoters which specifically confer superior abilities to degrade TCE upon certain microorganism host cells used in this invention. This system readily permits manipulation of the isolated genes by cloning into a variety of cloning vectors and expression via a variety of different promoter systems, so as to increase and optimize metabolic activity to degrade TCE. In addition, this system readily permits the study and manipulation of the specific enzymes and proteins involved in TCE degradation. As a consequence of the preparation of DNA segments containing PmKRl toluene monooxygenase genes, the incorporation of such DNA segments into suitable plasmid vectors and the transformation of microorganism host cells, PmKRl toluene monooxygenase enzyme products are expressed and may be isolated. Thus, a different approach to the degradation of TCE may be taken using isolated and purified enzyme products, rather than the transformed microorganism host cells. It is contemplated that the PmKRl toluene monooxygenase enzyme products could be used directly to degrade TCE. Such enzyme products could be released into or applied to loci of TCE chemical waste and could be useful in pollution control, for example, in the treatment of industrial waste water contaminated with TCE.
II. Description of the Art
Many different methods have been proposed for rendering toxic wastes innocuous. Among these are incineration, chemical transformation, and microbiological degradation. Because microbiological degradation of toxic waste does not involve the use of chemical reagents which might themselves be toxic and does not result in the production of large amounts of noxious fumes, such as produced in the incineration of toxic waste, it has become a preferred method of disposing of toxic waste.
Most microbiological degradations of toxic materials are based upon discovering a particular microorganism which will metabolize the toxic material, converting it to innocuous metabolic products, usually, in the case of toxic organic compounds, converting such compounds into carbon dioxide, water and salts. Finding microorganisms, in particular, genetically engineering microorganisms which can efficiently and safely convert toxic wastes into innocuous metabolic products is a highly complex procedure involving many arduous steps and requiring a significant expenditure of time. Most efforts thus far have focused on finding microorganisms indigenous to and isolated from contaminated soil or water.
One approach is to obtain a soil or water sample and enrich the sample for a mixture of microorganisms or isolate from a mixture a purified culture of a microorganism with the ability to degrade one or more toxic compounds. Several studies using mixtures of microorganisms containing methane-utilizating bacteria obtained by methane enrichment of a soil sample have shown that such mixtures have the ability to degrade TCE and other chlorinated ethenes. Fogel et al., Appl. Environ. Microbiol. 51: 720-724 (1986); Wilson and Wilson, Appl. Environ. Microbiol. 49: 242-243 (1985). Although these methane-utilizing cultures contain more than one type of bacterium, it is proposed that the methanotrophs are responsible for the degradation of TCE. Fogel et al. (supra) report a rate of TCE degradation of 0.03 nanomoles of TCE per minute per milligram of cell protein.
Other studies have not used mixtures of microorganisms but purified strains isolated from soil or water. One such approach is described in U.S. Pat. No. 4,493,895, wherein is claimed a process of microbial degradation of contaminating halogenated aromatic compounds into innocuous materials. This process comprises the steps of (1) collecting a sample of material from the site contaminated with obnoxious chemicals; (2) enriching the microorganisms found living in the sample; (3) separating the strains of microorganisms capable of having different metabolisms for the various chemicals in the sample from the site, from each other; (4) purifying the strains which are capable of biodegrading the chemicals to be disposed of; (5) applying the strain to the locus of the contaminants to be disposed of; and (6) monitoring of removal of the contaminants at the locus of the application. U.S. Pat. No. 4,477,570 describes and claims the microorganisms used in the above-described claimed process of U.S. Pat. No. 4,493,895.
Another approach is described in U.S. Pat. No. 4,664,805, wherein is claimed a process for decontaminating environments with halogenated organic compounds utilizing (1) microorganisms indigenous to the environment to be decontaminated which can metabolize but cannot grow on the contaminant; (2) a inoculum of microorganisms not indigenous to the environment which metabolize the contaminant faster than the indigenous microorganisms but cannot grow on it; and (3) a non-toxic analog of the contaminant which serves as a substrate for growth of the indigenous and non-indigenous microorganisms. Reliance is placed on microorganisms already present in the environment, so-called indigenous microorganisms to accomplish the degradation. The degradation is enhanced by the non-indigenous microorganism.
Yet another approach described in U.S. Pat. No. 4,511,657 involves a process of treating chemical waste landfill leachates with activated sludge containing bacteria capable of metabolizing obnoxious organics present in the leachates. All of the above described approaches involve the use of microorganisms which are indigenous to or isolated from contaminated soil or leachates. The degradative enzymes needed for microorganisms to degrade halogenated organic compounds may be encoded by genes borne on plasmids. A single plasmid generally contains genes encoding enzymes in a single degradative pathway. Plasmids have been employed in methods for the biodegradation of certain chlorinated aromatic organic compounds, as illustrated by U.S. Pat. No. 4,535,061 (which describes plasmid-assisted breeding procedures for generating pure and mixed cultures of microorganisms capable of dissimilating environmentally persistent chemical compounds) and U.S. Pat. No. 4,664,805 discussed above.
Using plasmids from microorganisms that degrade halogenated aromatic organic compounds (A.T.C.C. 31939-31945) (which microorganisms were described in U.S. Pat. Nos. 4,477,570 and 4,493,895) European Patent Application 8511008.1 discloses the preparation of hybrid plasmids and transconjugates containing these plasmids. European Patent Application 8511008.1 teaches a process for producing a microorganism having specificity for biodegrading halogenated organic compounds which comprises the steps of: (1) separately culturing and maintaining (a) a broad spectrum microorganism selected from the group of ATCC 31945 ATCC 31941, ATCC 31942, ATCC 31940, ATCC 31943, ATCC 31944, ATCC 31939 and mutants thereof, and (b) a broad host range vector; (2) separately isolating the plasmid-DNA from (a) and (b) above; (3) separately purifying the plasmid-DNA from (a) and (b) above; (4) separately enzymatically restricting the purified DNA from (a) and (b) above; (5) combining the products of step (4) and enzymatically ligating the combined products; (6) transforming the products of the ligation, into a receptive microorganism such as E. coli or species of Pseudomonas: (7) selecting those transformants having the desired plasmid-DNA inserted into the vector; (8) conjugating the selected plasmid-DNA into a receptive host selected from the group of Pseudomonas, Klebsiella, Rhizobium, Agrobacterium, Escherichia with aid of a helper plasmid; and (9) selecting those transconjugants having the desired plasmid DNA.
Eleven transconjugates were disclosed and unexpectedly a majority of the transconjugates were found to utilize as their sole carbon source certain aliphatic halogenated organic compounds, specifically, tetrachloroethylene, ethylene dichloride, methylchloroform and TCE, whereas the progenitor microorganisms A.T.C.C. 31939-31945 utilized only a broad spectrum of aromatic organic compounds as disclosed in U.S. Pat. No. 4,493,895. The only assay for utilization of these aliphatic halogenated organic compounds was growth on medium containing the compound where the growth was one-half greater than mean growth. Except for this growth assay, the transconjugates are completely uncharacterized. In particular, nothing is disclosed about the extent of degradation of these aliphatic halogenated organic compounds by these microorganisms or about the nature and toxicity of the metabolic products. Nothing is taught or disclosed regarding what genes, gene segments, enzymes, proteins or protein products are involved in the transconjugates' ability to metabolize such aliphatic halogenated organic compounds, including TCE. In particular, the teaching of European Patent Application 851008.1 is limited to utilization of aliphatic halogenated organic compounds, including TCE, by plasmids selected from the group of microorganisms designated as A.T.C.C. 31939-31945.
With respect to TCE metabolism specifically, partial degradation of TCE by anaerobic organisms has been reported but metabolites of the degradation process include vinyl chloride and dichloroethylene which are similarly harmful as groundwater contaminants. Kleopfer et al., Environ. Sci. Technol., 19:277-280 (1985); Parsons et al., J. Am. Water Works Assoc., 76:56-59 (1984); Vogel & McCarty, Appl. Environ. Microbiol., 49:1080-1083 (1985). A recent report describes a naturally occurring bacterial isolate which is capable of degrading TCE under aerobic conditions. Nelson et al., Appl. Environ. Microbiol., 52:383-384 (1986); Nelson et al., Appl. Environ. Microbiol., 53:949-954 (1986). The microorganism designated Strain G4 requires phenol, toluene, o-cresol or m-cresol for TCE degradation. As characterized, Strain G4 (1) does not utilize the TOL pathway for toluene degradation; (2) does not appear to have the enzyme toluene dioxygenase, the first enzyme in the TOD pathway for toluene degradation, and (3) does not utilize the TMO pathway for toluene degradation. These three toluene degradative pathways (TOL, TOD. TMO) are summarized in U.S. patent application Ser. No. 177,631. Nelson et al. does not teach or disclose what genes, gene segments, enzymes, proteins or protein products are involved in Strain G4's ability to degrade TCE nor whether the genes involved are plasmid encoded or chromosomally encoded. Genetic engineering of the Strain G4 has not been reported.
More recently, Nelson et al., Appl. Environ. Microbiol., 54: 604-606 (1988) have tested the TCE-degradative ability of 6 microorganism strains capable of degrading naphthalene, biphenyl, phenol and toluene. Only 2 of the strains tested, Pseudomonas putida PpFl (PpFl) and Pseudomonas putida B5 (B5), degraded TCE. PpFl and B5 are toluene degrading strains, however a third toluene degrading strain Pseudomonas putida mt-2 (Pp mt-2) did not degrade TCE. Thus, it appears that not all toluene degrading strains are capable of degrading TCE.
Pp mt-2, which Nelson et al., supra, found could not degrade TCE, contains the pWWO plasmid, which plasmid codes for enzymes of the toluene degradation pathway known as TOL. PpFl, which Nelson et al., supra, found could degrade TCE, is known to contain enzymes of the toluene degradation pathway known as TOD. Wackett and Gibson, Appl. Environ. Microbiol., 54:1703-1708 (1988), have also recently shown that PpFl cells have the ability to degrade TCE. Under their culture and assay conditions, Wackett and Gibson, supra, found that approximately 50% to 60% of input TCE (20 .mu.M) was degraded. In contrast, at high TCE concentrations (320 .mu.M) no TCE degradation could be detected. The highest TCE degradation rate that they observed was 1.8 nanomoles per minute per milligram of cell protein.
The genes for the TOD pathway of PpFl are chromosomally encoded, in contrast to the plasmid encoded TOL pathway genes of Pp mt-2. Thus, it is not possible to predict whether genes involved in TCE degradation are chromosomally encoded or plasmid encoded. Studies by Nelson et al., supra, and Wackett and Gibson, supra, with mutants of PpFl defective for various components in the TOD toluene degradative pathway suggest that the ability of PpFl to degrade TCE is associated with toluene dioxygenase enzyme activity, which is the first enzyme in the chromosomally encoded TOD pathway. Studies by Nelson et al., supra, with Pp mt-2 showed that TCE degradative ability is not associated with any enzymes of the plasmid-encoded TOL pathway.
None of the microorganisms tested thus far for TCE degradative ability utilize the plasmid-encoded TMO pathway for toluene degradation. In addition, no microorganism has yet been genetically engineered to increase enzyme activity and TCE degradative ability. Furthermore, the use of any of the above described microorganism systems to degrade TCE has several associated problems. A first problem is that in order to degrade TCE, a degradative enzyme pathway (for example, the TOD pathway) must be induced in the microorganisms and the inducers that must be added to the TCE contaminated sample are hydrocarbons. A second problem is that since TCE (a substrate for the induced degradative enzymes) itself cannot be used by these microorganisms as a carbon source for cell growth, cells must be provided with another substrate (a cosubstrate) for growth. Such cosubstrates added to the TCE contaminated sample are hydrocarbons, such as toluene. These two problems are related to the practical problem that in order to degrade TCE in a contaminated sample such as an aquifer, it is not desirable to have to add hydrocarbons, such as toluene (as inducer and/or carbon source) because hydrocarbons like toluene are themselves environmentally toxic compounds. A third problem, closely related to the first two, is that in the above described systems where a hydrocarbon such as toluene acts as the inducer of and substrate for the degradative pathway enzymes (which enzymes both metabolize the hydrocarbon and degrade TCE). there is a competition between the hydrocarbon and TCE for the same enzyme system. Under conditions where the hydrocarbon concentration is in great excess over the TCE concentration, the hydrocarbon will compete more effectively for the enzyme system and delay TCE degradation. These three problems illustrate various aspects of what is termed the cosubstrate problem which occurs in the previously described inducible enzyme systems for TCE degradation.